Many conformational transitions transpire during the course of an enzymatic reaction. Characterization of the physical details of these dynamical processes is essential for understanding enzyme catalysis, protein/ligand interactions, and fluctuations in the protein energy landscape. To date, the limited number of studies performed has addressed these issues in enzymes or proteins of low molecular weight. The majority of enzymes are larger than non-catalytic proteins and there is evidence that the dynamic and energetic properties of large proteins are distinct from smaller ones, therefore current understanding may not apply to other systems. In addition, the proper assessment of the interplay between function and dynamics requires a departure from the restraints of a single experimental technique. Accordingly, the role of dynamics and function will be addressed using solution NMR spectroscopy, ligand synthesis, biochemical characterization, and computation. The studies described in this proposal focus on large enzymes, VanX (46 kDa) an enzyme essential for bacterial resistance to the antibiotic vancomycin and triosephosphate isomerase (TIM, 54 kDa), a model enzyme whose dysfunction is associated with nonspherocytic hemolytic anemia and neurological disorders. The structure, dynamic, and energetic contributions to catalysis and stability will be addressed through studies of VanX and TIM in complexes that mimic discreet steps in the reaction pathway. The specific long-term goals of this application are to determine the structure and conformational changes in VanX that lead to inhibition of its catalytic activity and to its substrate preference for hydrolysis of amides over esters. For TIM, the proper timing of motional events for optimal catalysis will be investigated by NMR spin-relaxation measurements, site-directed mutation, and computational studies. This combination of experiments will allow characterization of the dynamics of catalytically important residues and the active site loop.